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. 2014 Jul 22;6(7):2162–2176. doi: 10.3390/toxins6072162

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© 2014 by the authors; licensee MDPI, Basel, Switzerland.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

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TcdA CROPs interact with TcdA^1–1874. (A) TcdA/TcdB are multi-domain toxins with a glucosyltransferase domain (GTD), cysteine protease domain (CPD), a hydrophobic region (HR), an intermediate domain (IMD) and the CROPs; (B) Glutathione-S-transferase (GST) (mock) or GST-TcdA CROPs were coupled to GSH beads and used as bait to precipitate CROP-truncated TcdA^1–1874. Coomassie staining proves equal applied amounts of GST and GST-TcdA CROPs (left panel). Immunoblot with α-TcdA^1–1065 shows the input and precipitation of truncated TcdA (right panels); (C) Binding of intermediate TcdA^1102–1847, the glucosyltransferase domain, TcdA^1–542, and the CROP-deletion mutant, TcdA^1–1874, to GST-fused TcdA CROPs was analyzed. Immunoblot targeting GST reflects equal amounts of bait (upper panel); immunoblot against the Penta-His tag displays the input and bound fraction of the N-terminal toxin fragments (lower panel).